The HIV-1 virion infectivity factor (Vif) is an essential regulatory protein required for HIV- 1 replication in natural target cells such as CD4+ T-cells and macrophages. Recent studies from our group and others have demonstrated that Vif is a critical viral counter defense that specifically neutralizes an innate antiviral defense mechanism, mediated by human APOBEC3G (A3G) and related cytidine deaminases. These antiviral proteins induce lethal modification of cytosines to uracils in newly synthesized minus-strand viral DNA, resulting in the production of non-functional viruses in the absence of the retroviral Vif protein. Vif hijacks cellular Cullin5 (Cul5), ElonginB, and ElonginC to form a viral E3 ubiquitin ligase that targets A3G for polyubiquitination and degradation. We have identified N,N,N',N'-Tetrakis-(2-pyridylmethyl)-Ethylenediamine (TPEN) as a small molecule inhibitor of Vif. The overall goal of this project is to further characterize the novel small molecule Vif inhibitor and its derivatives that restore the potent innate antiviral defense. In particular we propose 1. To examine the effect of TPEN on Vif functions from multiple HIV-1 subtypes as well as HIV-2. We have observed that TPEN is a potent inhibitor of HIV-1 Vif (NL4-3) function which allows the anti-viral cytidine deaminase A3G to suppress even the wild-type HIV-1 infectivity. It is not clear whether TPEN could inhibit Vif molecules from a wide range of HIV-1 subtypes. We will construct expression vectors for all the major subtypes of HIV-1 and HIV-2 Vif molecules and test the effect of TPEN on these Vif molecules. We will also determine whether TPEN could inhibit HIV-1 replication in primary CD4+ T cells which express endogenous A3G. Studies proposed in this aim are designed to further substantiate the effect of TPEN anti-HIV activity. 2. To examine the effect of derivatives of TPEN on Vif function and the anti-viral activity of A3G. We have observed that TPEN is a potent inhibitor of HIV-1 Vif function which allows the anti-viral mammalian cytidine deaminase APOBEC3G (A3G) to suppress even the wild-type HIV-1 infectivity. It is not clear whether TPEN inhibits Vif solely through zinc chelation, since another membrane permeable zinc chelator, chloroquinol, was not effective. To address this issue, we will evaluate TPEN derivatives that do not have the ability to bind zinc for anti-Vif activity. TPEN derivatives that have altered hydrophobic, hydrophilic, or charged properties will also be evaluated for their anti-Vif function. Studies proposed in this aim are designed to further characterize the mechanism of TPEN anti-HIV activity and to identify more potent anti-Vif inhibitors with a broad therapeutic window. PUBLIC HEALTH RELEVANCE: The HIV-1 virion infectivity factor (Vif) is an essential regulatory protein required for HIV- 1 replication in natural target cells such as CD4+ T-cells and macrophages. Recent studies from our group and others have demonstrated that Vif is a critical viral counter defense that specifically neutralizes an innate antiviral defense mechanism, mediated by human APOBEC3G (A3G) and related cytidine deaminases. We have identified a small molecule inhibitor of Vif. The overall goal of this project is to further characterize the novel small molecule Vif inhibitor and its derivatives that restore the potent innate antiviral defense.